Development of an improved tuberculosis (TB) vaccine is a high worldwide public health priority. Bacillus Calmette-Guerin (BCG), the only licensed vaccine for TB, is inadequate for TB control with provision of partial protection from disseminated forms of childhood TB and variable efficacy against adult pulmonary TB. Non-tuberculous mycobacteria (NTM) are a group of over 140 Mycobacterial species, which are found worldwide often in soil and water reservoirs. Some evidence suggests that NTM exposure varies substantially in different geographic regions and might explain the disparate outcomes of BCG vaccination, as well as susceptibility to Mtb infection and TB disease. Evidence supporting this hypothesis is increasing, although it has not been rigorously tested due to a lack of reliable tools to measure NTM-specific immune responses in individuals. Mycobacterium avium complex (MAC) species are the most common NTM that colonize and infect humans. We hypothesize that conserved epitopes exist between MAC and M. tuberculosis (Mtb) which induce heterologous T-cell responses during MAC infection that modulate immunity to Mtb and to TB vaccines. This study will develop the necessary reagents and assays to measure MAC-specific immune responses in clinical specimens and examine whether MAC infection modulates susceptibility to TB. In addition, NTM-specific assays could enhance and facilitate diagnoses, decrease invasive interventions in clinical care, and decrease costs. We will focus our initial efforts on MAC because it is the most common pathogenic NTM species. We hypothesize that MAC-specific T- cell responses are present in individuals with MAC infections (disease or colonization) and may be identified and developed into peptide reagents suitable for clinical assessment. To accomplish our goals, we have a collaborative partnership with complementary expertise in T-cell epitope discovery, human TB immunology, and clinical infectious diseases. This is an opportune time to develop and employ validated tools, which will enable us to examine questions regarding the influence of NTM exposure on the success of new TB vaccines.